Anticancer Compound

ABSTRACT

Azadirachta indica  cell suspension culture is used to metabolize dianabol to yield potent anticarcinogenic 17β-hydroxy-17α-methyl-5α-androst-1-en-3-one, which can also be synthesized.

BACKGROUND

Azadirachta indica cell suspension culture can be used for thebiotransformation of dianabol (Compound 1) to yield metabolitesincluding 17β-hydroxy-17α-methyl-5α-androst-1-en-3-one (Compound 2), and17β-hydroxy-17α-methyl-5α-androstan-3-one (Compound 3); these can bealternately synthesized chemically. The structures of these compoundswere deduced on the basis of various spectroscopic techniques.

Compound 2 exhibited a significant immunomodulatory inhibitory activityand strongly suppressed the PHA-activated T-cell proliferation (IC₅₀:<10.33 μM) comparable to control drug prednisolone, after 72 hoursincubation, and was further found to interfere with the IL-2 production(IC₅₀: 16.89±1.32) (FIG. 2A). Compound 2 also exhibited anticanceractivity against lung cancer cell line; NCI-H460, it moderatelyinhibited the growth of cancer cells (22.5±4.15 μM).

Compound 3 exerted a moderate inhibitory activity on both tests ascompared to Compound 2. On the other hand, the Compound 1 did not showany significant effect on the tested system.

Molecular docking studies were also performed to speculate possibleinteraction among IL-2 protein, and biotransformed products; studiesexhibited a good correlation between the predicted binding energies ofthe compounds acquired by the FlexX program and the experimentalaffinities. For docking studies, crystal structure of human IL-2complexed with Compound 4[(R)—N-[2-[1-(aminoiminomethyl)-3-piperidinyl]-1-oxoethyl]-4-(phenylethynyl)-L-phenylalaninemethyl ester] was downloaded from Protein data bank (pdb id: 1M48).

SUMMARY OF THE INVENTION

Plant cell suspension cultures can serve as tools for the in vivoproduction of secondary metabolites (1,2), as well as for thebiotransformation of xenobiotics (3-5). These cultures are considered tobe useful biocatalysts for reactions, such as hydroxylation at allylicpositions, oxidation-reduction between alcohols and ketones, and thereduction of carbon-carbon double bonds (6,7). Plant cellculture-mediated biotransformations are now increasingly employed bysynthetic chemists for the structural modifications of various organiccompounds in addition to using standard synthetic techniques.

Plant enzyme biocatalysts may be applied to the production of totallynew drugs and also may be used to modify existing drugs by improvingtheir bioactivity spectrum. The introduction of a functional group intoterpenoids, and steroids is an important reaction in syntheticchemistry. Many studies have been reported on the specific oxidation,reduction of olefins, and alicyclic hydrocarbons with chemical reagents(8-10). The ability of cultured plant cells to transform organiccompounds is useful for mass production of substances; however, chemicalsynthesis of these compounds remains a possibility. Plant cell culturesand microbacteria are considered to be useful biocatalysts for reactionssuch as the hydroxylation at allylic positions, the oxidation-reductionof alcohols and ketones, and reduction of carbon-carbon double bonds(11).

In the instant invention, biotransformation of Compound 1 was obtainedby incubating it with Azadirachta indica cell suspension cultures(12-13). This yielded Compounds 2 and 3, resulting from the sequentialreduction of olefinic double bonds. The structures of transformedcompounds were deduced by various spectroscopic methods.

DETAILED DESCRIPTION OF THE INVENTION

Compound 1, a potent steroid, is a derivative of testosterone,exhibiting strong anabolic and moderate androgenic properties. ThisCompound was first made available in 1960, and it quickly became themost favoured and widely used anabolic steroid by athletics.

Human health is directly influenced by the immune systems, and theirperformance, which are fundamentally designed for the protection againstthe attack of foreign invaders. However the onset of almost allinfectious and degenerative diseases is largely due to inadequate orhyperactive immune response. Therefore, the modulation of the immunesystem is highly relevant to the control of numerous immunologicaldisorders.

The instant invention reports the immunomodulatory actions of Compound2, which is found capable of reducing the PHA dependant Th1 responseinduced in human peripheral mononuclear cells.

The molecular characteristics of Compounds 1-3, possibly associated withthe inhibition of IL-2 protein, were also performed using modellingstudy involving the FlexX program to dock the compounds 1-3, along withthe reference inhibitor Compound 4, into the active site of the IL-2protein.

Usually docking protocol is evaluated through re-docking process inwhich the co-crystallized Compound is extracted and re-docked into thebinding cavity, and the quality of docking protocol is evaluated throughroot mean square deviation (RMSD ≦2 Å is considered as best). In thisinvention, Compound 4 was used as a reference for docking studies.Compound 4 synthesis has been reported by Tiley et at (27) and itschemical characterization is reported. The Compound was co-crystallizedwith IL-2 by Arkin et at (28). The re-docking of Compound 4, suggestedthat the docking protocol is suitable for the docking analysis of newlyidentified IL-2 inhibitors (Compounds 1-3).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Biotransformation of dianabol (Compound 1) by cell suspensioncultures of Azadirachta indica, and resulting metabolites, Compounds 2and 3.

FIG. 2: Effect of Compounds 1-3 on phytohemagglutinin (PHA) T-cellproliferation and production. (2A) The bar graph represents effects ofvarious concentrations of the test Compounds 1-3 after 72 h incubationwith peripheral blood mononuclear cells at 37° C. Effect of compounds onT-cell proliferation response is compared with non-proliferated (+ve)and proliferated (−ve) cells. The bar graph (2B) represents effects ofvarious concentrations of the test Compounds 1-3 on production of IL-2compared with (+ve) and without (−ve) PHA induced IL-2 production. Eachbar represents the mean value of triplicate reading ±SD.

FIG. 3: Chemical structure of Compound 4.

BIOTRANSFORMATION STUDIES

Biotransformation of Compound 1 by cell suspension culture ofAzadirachta indica A Juss, yielded compounds 2 and 3, (FIG. 1).

FIG. 1

Compound 2 was obtained as a colorless solid. The molecular formulaC₂₀H₃₀O₂ was deduced from the HREI-MS at m/z 302.0122 (Calculated at302.0120), indicating six degrees of unsaturation. The IR spectrumshowed hydroxyl absorption at 3445, and carbonyl absorption at 1674cm⁻¹. The ¹H NMR spectrum of Compound 2 displayed only one olefinicdouble bond at δ_(H) 7.13, and 5.83 (each d, J_(1,2)=10.2 Hz) (Table-1).

TABLE 1 ¹H NMR (300 MHz, CDCl₃)^(a)) chemical shifts of Compound 1 andits metabolites 2 and 3. δ in ppm and J in Hz. Com- pound NO 1 2 3  17.03 (d, J_(1,2) = 10.2) 7.13 (d, J_(1,2) = 10.2) 2.05-2.08 (m)^(b)  26.19 (dd, J_(2,1) = 10.2, 5.83 (d, J_(2,1) = 10.2) 1.68-1.70 (m)  3 — ——  4 6.04 (br. s) 2.20-2.23 (m) 2.06-2.08 (m)^(b)  5 — 1.92-1.94 (m)1.93-1.95 (m)  6 2.32-2.34 (m) 2.28-2.30 (m) 2.30-2.32 (m)  7 1.28-1.30(m) 1.32-1.35 (m) 1.30-1.32 (m)  8β 1.67-1.70 (m)^(c) 1.50-1.52 (m)1.47-1.50 (m)  9α 1.00-1.02 (m) 1.15-1.18 (m) 0.92-0.95 (m) 10 — — — 111.62-1.64 (m)^(b) 1.63-1.65 (m)^(b) 1.51-1.53 (m) 12 1.20-1.22 (m)1.21-1.23 (m) 1.18-1.20 (m)^(c) 13 — — — 14α 1.15-1.18 (m) 1.12-1.14 (m)1.18-1.20 (m)^(c) 15 1.30-1.32 (m) 1.41-1.44 (m)^(c) 1.36-1.38 (m) 161.68-1.70 (m)^(c) 1.71-1.73 (m) 1.64-1.66 (m) 17 — — — 18 0.91 (s) 0.87(s) 0.85 (s) 19 1.22 (s) 1.01 (s) 1.01 (s) 20 1.16 (s) 1.20 (s) 1.19 (s)^(a))assignments based on COSY and HMQC; ^(b,c)signals may beinterchanged.

The ¹³C NMR spectra also showed disappearance of one olefinic doublebond in the Compound 2. It showed additional signal of C-4 methylene(δ_(C) 41.0), and C-5 methene (δ_(C) 44.4) (Table-2). This reductionalso confirmed by the HMBC spectrum, in which C-1 (δ_(H) 7.13) have J₂correlations with C-2 (δ_(C) 127.5), and C-10 (δ_(C) 39.1), and J₃correaltions with C-3 (δ_(C) 200.1) and C-5 (δ_(C) 44.4). Similarly, C-5(δ_(H) 1.92-1.94, m) showed J₂ correlations with C-4 (δ_(C) 41.0), andC-10 (δ_(C) 39.1), and J₃ correlation with C-3 (δ_(C) 200.1). Theconfiguration of the newly introduced proton at C-5 was assigned to be aon the basis of NOESY correlation between H-5 with CH₃-20α, which showeda trans junction between rings A and B. Thus, the structure of Compound2 was identified as 17β-hydroxy-17α-methyl-5α-androst-1-en-3-one (14).

Compound 3 was also obtained as a white solid. The HREI-MS of Compound 3showed the M⁺ at m/z 304.2113, corresponding to the formula C₂₀H₃₂O₂(Calculated at 304.2115). The IR spectrum showed a hydroxyl absorptionat 3360, and ketonic absorption at 1713 cm⁻¹. The reduction of botholefinic double bonds was inferred by the absence of the all downfieldolefinic protons in the ¹H NMR spectrum of Compound 3 (Table-1). The ¹³CNMR spectra of Compound 3 also showed three additional upfield methyleneand a methine carbon signals i.e. C-1 (δ_(C) 38.2), C-2 (39.0), C-4(44.7) and C-5 (δ_(C) 46.8), (Table-2). In the HMBC spectrum, C-19methyl protons (δ_(H) 1.01) showed J₂ correlations with C-10 (δ_(C)35.8), and J₃ correlations with C-1 (δ_(C) 38.2), and C-5 (δ_(C) 46.8).The configuration of the newly introduced proton at C-5 was assigned tobe a on the basis of NOESY correlation between H-5 with CH₃-20α, whichsupported a trans junction between rings A and B.¹⁵ The complete ¹H and¹³C NMR assignments of Compound 3 are presented in Tables-1 and -2,respectively.

TABLE 2 ¹³C NMR (100 MHz, CDCl₃)^(a))^(b)) chemical shifts of Compound 1and its metabolites 2 and 3. C. NO. 1 2 3 1 155.7 (d)  158.3 (d)  38.2(t) 2 127.5 (d)  127.5 (d)  39.0 (t) 3 186.2 (s)  200.1 (s)  211.9 (s) 4 123.8 (d)  41.0 (t) 44.7 (t) 5 169.0 (s)  44.4 (d) 46.8 (d) 6 32.8 (t)27.6 (t) 28.9 (t) 7 33.3 (t) 31.6 (t) 31.6 (t) 8 36.4 (d) 36.6 (d) 36.2(d) 9 52.5 (d) 50.7 (d) 53.9 (d) 10 43.6 (s) 39.1 (s) 35.8 (s) 11 22.5(t) 20.9 (t) 21.1 (t) 12 31.3 (t) 31.0 (t) 31.5 (t) 13 45.6 (s) 45.7 (s)45.6 (s) 14 49.8 (d) 50.1 (d) 50.6 (d) 15 23.3 (t) 23.2 (t) 23.3 (t) 1638.7 (t) 39.0 (t) 38.6 (t) 17 81.3 (s) 81.5 (s) 81.6 (s) 18 13.9 (q)14.1 (q) 14.0 (q) 19 18.7 (q) 13.1 (q) 11.5 (q) 20 25.8 (q) 25.9 (q)25.8 (q) ^(a))multiplicities were determined by DEPT experiments;^(b))assignment based on HMQC and HMBC.

Biological Studies Anti-Inflammatory:

In this invention, effects of Compounds 2 and 3 on the innate immuneresponse, in particular the reactive oxygen species (ROS) production wasexamined using whole blood phagocytes, and isolated neutrophils, whichdid not result in any significant effect (Table 3). In addition to that,effects of these compounds on T-cells proliferation were also evaluated;investigating their (Compounds 2 and 3) ability to modulate PHAactivated T-cell proliferation response and production of IL-2 cytokine.

Compound 2 was found to have significant inhibitory activity on T-cellproliferation with IC₅₀ value less than 10.33 μM compared to Compound 1,which did not show any significant effect neither on T-cellsproliferation nor IL-2 production. A moderate inhibitory activity withIC₅₀ value of 42.11 μM was obtained with Compound 3. The activity ofthese Compounds 2 and 3 was further confirmed by their effects on IL-2cytokine production, which is the main contributor in T-cell activation.The extracellular production of IL-2 from peripheral blood mononuclearcells was significantly inhibited (IC₅₀=16.9±1.32 μM) by Compound 2. Onthe other hand, Compound 3 was having a moderate inhibitory effect(IC₅₀=49.3±1.32 μM) on IL-2 production (FIG. 2B). The activity ofCompound 2, in contrast to the 1 and 3, could be due to the olefinicbond between C-1/C-2. Compound 3 did not possess a carbon-carbon doublebond, and showed lesser activity (Table-3). This suggested the activitymight be due to C-1/C-2 carbon-carbon double bond.

TABLE 3 Comparative IC₅₀ effect of Compound 1 and its transformedmetabolites, Compounds 2 and 3 on oxidative burst of whole blood,isolated polymorphoneutrophils (PMNs), T-cell proliferation and IL-2cytokine Oxidative burst IC₅₀ T-Cell (μM) on proliferation IC₅₀ (μM)Comp. Code Whole blood PMNs IC₅₀ (μM) IL-21 >333.33 >166.67 >166.67 >166.67 2 >331.13 >165.56  <10.33 16.89 ± 1.323 >328.95 >164.47 42.11 ± 11.51 49.34 ± 1.32

Anti-Cancerous:

Compounds 1-3 were also screened for the anticancer activity againstNCI-H460 cell line. Compound 2 gave the best results having GI₅₀ value22.5±4.15 μM which means, at this concentration this Compound inhibited50% growth of the respective cell line i.e., NCI-H460 (Table 4).Comparing the GI₅₀ values of Compounds 1 and 3, this is the lowesteffective concentration of the three compounds used against NCI-H460.Compounds 1-3 have similar structures (FIG. 1) except for one and twooleifinic double bonds. Compound 2 differs by absence of one and 3 bytwo oleifinic double bonds. Interestingly, Compound 2 has a differenceof only one oleifinic double bonds at C-4 position as compared to itsparent compound; probably due to the absence of this double bondCompound 3 was not able to inhibit considerable growth of cancer cellsat 100 μM that is why this Compound was not evaluated further. AndCompound 1 (substrate), which is different having two oleifinic doublebond (at C-1 and C-4 positions) exhibited better growth inhibition butnot as discriminating as Compound 2, which clearly suggested thatabsence and presence of oleifinic double bond in the given structures,is important for having the anticancer activity. Particularly, doublebond present at position C-4 is more important than at C-1. ConsequentlyCompounds 1 and 2 showed anticancer activity but Compound 3 wasnon-active against NCI-H460 cell line.

TABLE 4 Growth inhibition induced by transformed compounds against humanlung cancer cell line (NCI-H460). Compound(s)* GI₅₀ (μM) TGI (μM)doxorubicin 0.08 ± 0.02 — (control) 1 28.3 ± 2.72 ND 2 22.5 ± 4.15 60 ±9.2 3 ND ND ND: Values could not be determined indicates <50% inhibitionof cell growth at maximum dose (100 μM) tested.

Docking Studies

Docking studies showed a good correlation between the predicted bindingenergies of the compounds obtained by the FlexX program, and theexperimental binding affinities (Table-5).

TABLE 5 Docking Energies and H-Bond Distances with the Arg38. CompoundsBinding Scores H-bond Distance (Å) 4 −26.0 Arg38 3 −9.8 3.207 (NH2 

 ¹) 2 −10.8 1.820 (NH2 

 ¹) and 2.962 (NH2 

 ²) 1 −6.7 —

Analysis of the docking results revealed that all three compounds bindat the same receptor site on the surface of IL-2 protein. Comparing thebinding scores of the Compounds 1-3, with that of the reference ligand,it was predicted that Compounds 1-3 could inhibit IL-2 protein. Allthree compounds showed similar binding pattern and interacts on theactive site of IL-2 protein at the surface through several active-siteamino acid residues.

Compound 1 was the least active due to the presence of two olefinicdouble bonds in conjugation with the carbonyl group of ring A at C-3.The lone pair of the carbonyl group is least likely to be available tointeract with surrounding amino acid residues due to conjugation. Whilein Compound 2, the carbonyl group of ring A interact with NH2

of Arg38 with a weak hydrogen bond (3.207 Å), which is due to the mostsuitable geometry of ring A.

Olefinic double bond at C-1 is the difference between Compounds 2 and 3,and Compound 3 lacks any olefinic double bond. This structuraldifference makes the Compound 2 most active. The carbonyl group of ringA at C-3 position interact with the side chain amino groups of Arg38,and create two hydrogen bonds at a distance of 1.8 and 1.9 Å with NH2

and NH2

of Arg38, respectively. All three compounds are additionally stabilizedby a number of hydrophobic interactions with the active site aminoacids, notably Lys35, Arg38, Met39, Thr41, Phe42, Lys43, Phe44, Pro65,Val69, Leu72, and Ala73 at the A′B loop of the IL-2.

The docking results revealed the importance of Arg38 in the vicinity ofcarbonyl group of ring A, which plays a vital role in protein-ligandcomplex formation and stabilization.

Experimental General Methods

The ¹H-NMR spectra were recorded in CDCl₃ on Bruker AM-300 and AM-400NMR spectrometers with TMS as an internal standard using UNIX operatingsystem at 300 and 400 MHz, respectively. The ¹³C-NMR spectra wererecorded in CDCl₃ at 100 MHz on Bruker AM-400 NMR spectrometer. HREI-MSwere recorded on Jeol JMS 600 and HX 110 mass spectrometers with thedata system DA 5000. The IR spectra were recorded on a Jasco A-302spectrophotometer. The UV spectra were recorded on a Hitachi U-3200spectrophotometer. The optical rotations were measured on JASCO DIP-360digital polarimeter. The melting point was determined on a Buchi 510apparatus. Column chromatography (CC) was carried on silica gel column(70-230 Mesh). Purity of the samples was checked by TLC on pre-coatedsilica gel GF-254 preparative plates (20×20 cm, 0.25 mm thick, Merck)and were detected under the UV lights (254 and 366 nm), while cericsulphate was used as spraying reagent. Dianabol (Compound 1) waspurchased from Fluka Riedel-deHaën®.

Callus Culture

The callus cultures of the plant were derived from young leavescultivated in 300 mL jars, each having 25 mL of Murashige and Skoogmedia (16), supplemented with sucrose (30 g/L), 3-indole butyric acid(IBA) (19.7 μM), and 6-benzyl aminopurine (BA) (4.44 μM), and solidifiedby agar (6 g/L) at 25±1° C. under complete darkness.

Biotransformation Protocol

Cell suspension cultures were derived from static cultured cells inErlenmeyer flasks (1,000 mL), each containing 400 mL of the Murashigeand Skoog media, supplemented with ingredients as mentioned above,except BA and agar. After 15 days of pre-culturing on a gyratoryplatform shaker at 100 rpm, and with a 16 h photoperiod at 25±1° C., asolution of substrate (100 mg in 1 mL of acetone) was added to eachflask through a 0.2 μM membrane filter, and the flasks were placed on ashaker for 20 days. Taking aliquots from culture on daily basis carriedout the time course study and TLC was used to analyse the content oftransformation. A negative control containing only plant cell suspensioncultures, and a positive control-containing Compound 1 in the media werealso prepared in order to check the presence of plant metabolites in thecell culture, and the chemical changes as a result of chemical reaction(if any) due to media components, respectively.

Extraction and Isolation Procedure

After 20 days of incubation, the cells and the media were separated byfiltration. The filtrate was extracted with CH₂Cl₂ (3×1.5 L) and thecells were extracted in an ultrasonic bath with CH₂Cl₂ (3×500 mL) atroom temperature. The combined extract were dried over anhydrous Na₂SO₄,and concentrated under reduced pressures, which afforded a brown residue(1.32 g). The transformed metabolites were isolated from this gummycrude by using repeated column chromatography (silica gel) withpetroleum ether/EtOAc gradient, afforded metabolites 2 (26 mg, petroleumether:EtOAc, 9.5:0.5, 5.2% yield) and 3 (18 mg, petroleum ether:EtOAc,9.8:0.2, 3.6% yield).

17β-Hydroxy-17α-methyl-5α-androst-1-en-3-one (Compound 2)

Colorless solid, m.p. 140-142° C., [α]_(D) ²⁰ −40° (c 0.04, CHCl₃). UV(MeOH) λ_(max) (log ε): 230 nm (3.62). IR (CHCl₃) ν_(max): 3445 (OH),1674 (C═O), 1652, 1496 (C═C), 1380 cm⁻¹ (C—O). EI-MS m/z (rel. int. %):302 (10), 284 (3), 245 (15), 232 (3), 200 (5), 160 (18). HREI-MS m/z:302.0122 (M⁺, C₂₀H₃₀O₂; calcd 302.0120). ¹H(CDCl₃, 300 MHz) and ¹³C NMR(CDCl₃, 100 MHz) data listed in Tables-1 and -2, respectively.

17β-hydroxy-17α-methyl-5α-androstan-3-one (Compound 3)

Colorless solid, m.p. 152-154° C., [α]_(D) ²⁰ −130° (c 0.02, CHCl₃). UV(MeOH) λ_(max) (log ε): 203 nm (0.43). IR (CHCl₃) ν_(max): 3360 (OH),1713 (C═O), 1372 cm⁻¹ (C—O). EI-MS m/z (rel. in %): 304 (20), 289 (28),271 (15), 247 (45), 231 (39), 215 (14), 189 (12), 175 (16), 163 (46).HREI-MS m/z: 304.2113 (M⁺, C₂₀H₃₂O₂; calcd 304.2115). ¹H (CDCl₃, 300MHz), and ¹³C NMR (CDCl₃, 100 MHz) data listed in Tables-1 and -2,respectively.

[(R)—N-[2-[1-(Aminoiminomethyl)-3-piperidinyl]-1-oxoethyl]-4-(phenylethynyl)-L-phenylalaninemethyl ester] (Compound 4) Immunomodulatory Studies Reagents, Chemicals,and Equipment

Luminol (3-aminophthalhydrazide), Hanks Balance Salts Solution (HBSS)and Lymphocytes Separation Medium (LSM) were purchased from ResearchOrganics, Sigma, Germany, and MP Biomedicals, Inc., Germany,respectively. Zymosan-A (Saccharomyces cerevisiae origin),Dimethylsulfoxide (DMSO), ethanol and ammonium chloride of analyticalgrades were purchased from Merck Chemicals, Darmstadt, Germany. Theluminometer used was Luminoskan RS, Finland.

Isolation of Human Polymorphonuclear Cells (PMNs)

Heparinized blood was obtained by vein puncture aseptically from healthyvolunteers (25-38 years age). The buffy coat containing PMNs wascollected by dextran sedimentation and the cells were isolated after theLSM density gradient centrifugation from the tube base. Cells werewashed twice and suspended in Hank's Balance Salt Solution (Ca²⁺ andMg²⁺ free) (HBSS⁻), pH 7.4. Neutrophils were purified from RBCscontamination using hypotonic solution. Cells were adjusted to theirrequired concentration using Hank's Balance Salt Solution containingCa²⁺ and Mg²⁺ (HBSS⁺⁺).

Oxidative Burst Study

Luminol-enhanced chemiluminescence assay was performed as described byHelfand et al. (17), and Haklar et al. (18) with some modifications.Briefly, 25 μL diluted whole blood (1:200 dilution in sterile HBSS⁺⁺) or25 μL of PMNs (1×10⁶) cells were incubated with 25 μL of seriallydiluted compounds with concentration ranges between 3.2-50 mg/mL. Testswere performed in white 96-well plates, which were incubated at 37° C.for 30 minutes in the thermostated chamber of the luminometer. Opsonizedzymosan-A, 25 μL, followed by 25 μL luminol (7×10⁻⁵M) along with HBSS⁺⁺was added to each well to obtain a 100 μL volume/well. Wells receivedHBSS⁺⁺, and cells but no compounds were used as a negative control.Chemiluminescence peaks were recorded with the Luminometer. Results weremonitored as chemiluminescence relative light units (RLU) with peak andtotal integral values.

T-Cell Proliferation Assay

The T-cell proliferation assay was performed as described by Nielson etal. (19). In this assay, isolated lymphocytes were stimulated by addingthe phytohemagglutinin (PHA) in culture. The rate of proliferation andsurvival was measured by radiolabelled thymidine incorporation method.The lymphocytes were isolated as described by Boyum (20). A 50 μL ofcell suspension (10⁶/mL) was added to each well of a 96-well roundbottom tissue culture plate. 50 μL of PHA was added in each well fromthe working solution (20 μg/mL) to have final concentration 5 μg/mL. Thefinal volume was adjusted to 200 μL in each well by adding complete RPMImedia. The plates were incubated at 37° C. for 72 hours (90% humidity,5% CO₂/air). Methyl-³H thymidine (Amersham Pharmacia Biotech) 0.5 μCiwas added in each well and incubated for further 18 hours. Cells wereharvested on a filter mat (Type G-7) using cell harvester (INOTECHIH-280, Switzerland) and the radioactivity (CPM) was measured using aβ-scintillation counter (LS 6500, Beckman Coulter, USA). (FIG. 2A)

FIG. 2 IL-2 Cytokine Production

IL-2 cytokine was produce from peripheral blood mononuclear cells(PBMC). IL-2 cytokine production by PHA activated cells in the presenceor absence of test compounds was studied by ELISA using the humancytokine kit (Diaclone, Besancon Cedex France). Briefly, freshlyprepared mononuclear cells (10⁵/well) were cultured in 96-wellmicrotiter flat bottom plate in the presence or absence of 5 μg/mL PHA.Four different concentrations (10.33-331.13 μM/mL) of compounds, alongwith PHA, were used in this assay. The culture plate was incubated at37° C. for 18 h. Then the supernatant was collected and analyzed forIL-2 cytokine production following kit manufacturer instructions.

Statistical Analysis

Students T-test was performed to compare the significance meandifferences between the control and tested extracts for variouschemiluminescence results. The differences were considered to besignificant at levels of P≦0.05.

Anticancer Activity

The sulforhodamine B (SRB) protein-staining assay was employed formeasurement of in vitro growth inhibition and cytotoxicity (21) Theappropriate cell density (cells/well) of NCI-H460 cells (1×10⁴) wasadded in 96-well plates. The cell density used was dependent on thedoubling time of the cell line leading to the formation of monolayer.After 24 h incubation, different doses of Compounds 1-3 were added andincubated for further 48 h. This was followed by fixation of cells withice-cold trichloroacetic acid (50 μl, 50%) at room temperature for 30min. The plates were carefully washed five times with distilled water,and left for overnight drying in air. Sulforhodamine B dye (100 μl, 0.4%in 1% acetic acid) was introduced in each well and after 30 min residualdye was removed using acetic acid (1%) and air-dried overnight. Thebound SRB dye was solubilized in Tris-base solution (100 μl, 10 mM) withgentle shaking on a plate-shaker for 5 min prior to optical density (OD)measurements at 545 nm in a plate reader. Drug concentrations causinggrowth inhibition (GI₅₀) of 50% cells were calculated from dose responsecurves and total growth inhibition of Compound 2 was also evaluated.

Molecular Docking Protocol

The protein data bank file, PDB id; 1M48 (2.0 Å structure resolution) ofthe Human Interleukin-2 complexed with Compound 4 was obtained from theprotein data bank [Research Collaboratory for Structural Bioinformatics(RCSBu) (http://www.rcsb.org/pdb)]. The three-dimensional structures ofcompounds 1-3 were generated by molecular modelling software SYBYL6.9.²² Energy minimization was carried out using the Tripos force fieldwith a distance gradient algorithm with a convergence criterion of 0.05kCal/(molÅ), and maximum 10,000 iterations, respectively, withGasteiger-H{umlaut over (ú)}ckel charges (23).

Docking of Compounds 1-3 into the active site of IL-2 receptor wasperformed using FlexX docking software implemented in SYBYL6.9 (23).FlexX software is a fast and flexible algorithm for docking small ligandinto binding sites of the enzymes, using an incremental constructionalgorithm that actually builds the ligand in the binding site (24). Thesoftware incorporates protein-ligand interactions, placement of theligand core, and rebuilding of the complete ligand. A receptordescription file (RDF) was created from the PDB coordinates. The activesite for docking was defined as all atoms within 6.5-Å radius of theco-crystallized Compound 4. The proposed interaction mode of the ligandin the active-site of IL-2 was determined as the highest scoredconformation (best fit ligand) among 30-generated conformations andbinding modes generated according to FlexX scoring function, which isthe structure with the most favorable free energy of binding. Dockingresults were analyzed by VMD (visual molecular dynamics) (25).

REFERENCE

-   1. Charlwood, B. V.; Rhodes, M. J. C. Secondary Products from Plant    Tissue Cultures; Clarendon Press: Oxford, 1990; pp 167-200-   2. Lowe, K. C.; Davey, M. R.; Power, J. B. Plant tissue culture:    past, present and future. Plant Tiss. Cult. Biotechnol. 1996, 2,    175-186.-   3. Reihard, E.; Alfermann, Fiechter, A. W. Advances in Biochemical    Engineering; Springer: New York, 1980; pp. 49-83-   4. Charlwood, B. V.; Hegarty, P. K.; Charlwood, K. A.; Lippin, G.;    Tampion, J.; Stride J. In Secondary Metabolism in Plant Cell    Cultures. Morris, P.; Scragg, A. H.; Stafford, A.; Fowler, M. W.,    Eds.; Cambridge University Press: London, 1986; pp 113-116.-   5. Dia, J.; Guo, H.; Lu, D.; Zhu, W.; Zhang, D.; Zheng J.; Guo, D.    Biotransformation of 2α, 5α, 10α, 14α, tetra acetoxy-(20), 11    taxadiene by ginkgo cell suspension cultures. Tettrahedron Lett.    2001, 42, 4677-4679.-   6. Suga, T.; Hirata, T. Biotransformation of exogenous substrates by    plant cell cultures. Phytochemistry, 1990, 29, 2393-2406.-   7. Ishihara, K.; Hamada, H.; Hirata, H.; Nakajima N.    Biotransformation using plant cultured cells. J. Mol. Catal. B:    Enzym. 2003, 23, 145-170.-   8. Mukaiyama, T.; Yamada, T.; Nagata, T.; Imagawa, K. Asymmetric    Aerobic Epoxidation of Unfunctionalized Olefins Catalyzed by    Optically Active α-Alkoxycarbonyl-β-ketoiminato Manganese (III)    Complexes. Chem. Lett. 1993, 2, 327-330.-   9. Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;    Hartung, J.; Jeong, K.; Kwong H.; Morikawa, K.; Wang, Z.; Xu, D.;    Zhang, X. The osmium-catalyzed asymmetric dihydroxylation: a new    ligand class and a process improvement. J. Org. Chem. 1992, 57,    2768-2771.-   10. Sakamaki, H.; Take, M.; Matsumoto, T.; Iwadare, T.; Ichinohe, Y.    Transformation of cycloartanyl acetate into B-homo triterpenoids. J.    Org. Chem. 1988, 53, 2622-2624.-   11. Sakamakia, H.; Itoha, K. I.; Taniaib, T.; Kitanakac, S.;    Takagid, Y.; Chaie, W. Horiuchie, C. A. J. Mol. Cat. B: Enzym. 2005,    32, 103-106.-   12. Choudhary, M. I.; Siddiqui, Z. A.; Khan, S., Saifullah;    Musharraf, S. G.; Atta-ur-Rahman. Biotransformation of    (−)-Caryophyllene Oxide by Cell Suspension Culture of Catharanthus    roseus. Z. Naturforsch, 2006, 61b, 197-200.-   13. Azizuddin; Saifullah; Khan, S.; Choudhary, M. I.;    Atta-Ur-Rahman. Biotransformation of Dydrogesterone by Cell    Suspension Cultures of Azadirachta indica. Turk. J. Chem. 2008, 32,    141-146.-   14. Hampel, V. B.; Kraemer, J. M. Die Kernresonanzspektren Von    Steroiden In Polaren Lösungsmitten II. Tetrahedron, 1966, 22,    1601-1613-   15. Thevis M.; Schänzer W. Mass Spectrometric Analysis of    Androstan-17β-ol-3-one and androstandiene-17β-ol-3-one isomers. J.    Am. Chem. Soc. for mass spectrum. 2005, 16, 1660-1669.-   16. Murashige, T.; Skoog, F. A revised medium for rapid growth and    biossays with tobacco tissue cultures. Physiol. Plant. 1962, 15,    473-497.-   17. Helfand, S.; Werkmeister, J.; Roder, J. Chemiluminescence    response of human natural killer cells. The relationship between    target cell binding, chemiluminescence, and cytolysis. J. Exp. Med.    1982, 156, 492-505.-   18. Haklar, G.; Ozveri, E. S.; Yuksel, M.; Aktan, A.; Yalynn, A. S.    Different kinds of reactive oxygen and nitrogen species were    detected in colon and breast tumors. Cancer Lett. 2001, 165,    219-224.-   19. Nielsen, M.; Gerwien, J.; Nielsen, M.; Geisler. C.; Ropke, C.;    Svejgaard, A.; Odum, N. MHC class II ligation induces CD58    (LFA-3)-mediated adhesion in human T cells. Exp. Clin. Immunogenet.    1998, 15, 61-68.-   20. Boyum, A. Isolation of mononuclear cells and granulocytes from    human blood. Isolation of mononuclear cells by one configuration,    and of granulocytes by combining centrifugation and sedimentation.    Scand. J. Clin. Lab. Invest. 1968, 21, 77-89.-   21. Skehan, P.; Streng, R.; Scudiero, D.; Monks, A.; McMahon, J.;    Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New    colorimetric cytotoxicity assay for anti-cancer drug screening. J.    Natl. Cancer Inst., 1990, 82, 1107-1112.-   22. SYBYL molecular modeling software. Tripos Associated Ltd., St.    Louis Mo.-   23. TRIPOS Inc., 1699 South Hanley Road, St. Louis, Mo. 63144, USA-   24. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. A fast flexible    docking method using an incremental construction algorithm. J. Mol.    Biol. 1996, 261, 470-489.-   25. Visual Molecular Dynamics Version 1.8.6, Theoretical and    computational Biophysics Group, University of Illinois & Beckman    Institute, 405 N. Matthews, Urbana, Ill. 61801.-   26. Jefferson W. Tilley, Li Chen, David C. Fry, S. Donald Emerson,    Gordon D. Powers, Denise Biondi, Tracey Varnell, Richard Trilles,    Robert Guthrie, Francis Mennona, Gerry Kaplan, Ronald A. LeMahieu,    Mathew Carson, Ru-Jen Han, C.-M. Liu, Robert Palermo, and Grace Ju,    Identification of a Small Molecule Inhibitor of the IL-2/IL-2Rr    Receptor Interaction Which Binds to IL-2 J. Am. Chem. Soc. 1997,    119, 7589-7590.-   27. Michelle R. Arkin, Mike Randal, Warren L. DeLano, Jennifer Hyde,    Tinh N. Luong, Johan D. Oslob, Darren R. Raphael, Lisa Taylor, Jun    Wang, Robert S. McDowell, James A. Wells, and Andrew C. Braisted,    Binding of small molecules to an adaptive protein-protein interface,    PNAS, 2003, 100, 1603-1608.

1. A method of treating lung cancer a therapeutically effective amountof 17β-hydroxy-17α-methyl-5α-androst-1-en-3-one.
 2. The method of claim1 wherein the mode of treatment includes administering a dosage formcontaining sufficient quantity of17β-hydroxy-17α-methyl-5α-androst-1-en-3-one and pharmaceuticaladjuvants.
 3. The method of claim 1 wherein17β-hydroxy-17α-methyl-5α-androst-1-en-3-one is obtained as abiotransformation product of dianabol contacted with a suspension ofAzadirachata indica cell culture under suitable conditions.